1088
BIOCHEMICAL SOCIETY TRANSACTIONS
(a)
Urine samples were first dcproteinized, using Millipore
PLGC membrane filters, and then 25 111 aliquots were
derivatized with phcnylisothiocyanate and evaporated to
dryness. T h e dried residue was taken u p in I00 pI o f which
1 0 pl was applied to the column. A typical chromatogram o f
urine is shown in Fig. I ( h ) , which shows that C M L had ii
retention time of 12.6 min under the conditions used (see
legend of Fig. 1 for details). T h e identification o f CMI. was
confirmed by demonstrating that the peak co-eluted with
authentic material.
Early morning urine samples were collected from 29 nondiabetic males with an age range from 4 to 60 years. C M L
and crcatinine were measured in thcsc samples. T h e mean
value fs.11 obtained was I 1. I f 7.3 (range 4.5CML/g of creatininc. There was no significant tre
In a separate study, using non-timed daytime samples,
values were lower, S . 8 f 1.9 mg of CML/g o f crcatinine
(range 4.2-9.1) in five non-diabetics and 8.6 f 3 . 9 mg o f
CML/g of creatininc (range 3.9- 13.6)in nine diabetics.
A simple, specific and sensitive assay for measuring C M L
in urine has been established. T h e values for C M L in urine
are in good agreement with earlier published values [ 21, but
arc about 10-fold greater than those obtained for fasting
urine samples 171. T h e implication o f this is that C M L
derived from dietary protein is a major contributor t o
urinary CML. This matter needs t o be clarified heforc furr studies can be performed. I t will then be possible t o
ess whether levels o f C M L arc affected by age and by
betes. A n alternative is t o measure C M L in tissue samples
after extraction and hydrolysis of long-lived proteins. It may
be that the relative rate o f formation of C M L comparcd to
advanced glycation end-products is reduced in patients who
are at greatest risk o f the chronic complications o f diabetes.
al
5
10
Elution time (min)
15
Fig. 1. H.p.1.c. chromatogrums showitig CML in (u) u
stundurd (0.0.7 mM) und (h) a urine sample
Aliquots ( 2 5 p l ) were derivatized with phenylisothiocyanatc,
vacuum dried and dissolved in 100 pl of 5 mwphosphatc
buffer, p H 7.4, containing 5% (v/v) acetonitrile. Ten microlitres was applied to a Pico-Tag column (15 cm x 3.9 cm)
maintained at 38°C. T h e column eluent was monitored at
254 nm. Two eluents A [70 mM-sodium acetate at pH 6.4
containing 6 % (v/v) acetonitrile] and B [60% (v/v) acetonitrile and 40% (v/v) methanol) were used to produce the
following gradient: time (min), %B, flow rate (ml/min); 0- 1 0 ,
0, I ; 10-10.5, 46, 1; 10.5-11.5, 100, I ; 1I.S-l2.0, 100, 1 ;
12.0-12.5, 100, 1.5; 12.5-20,0, 1.5; 20-20.5,0, 1 .
pre-column derivatization of amino acids with phenylisothiocyanate. Elution conditions were varied until a good separation was achieved. A typical chromatogram for C M L is
shown in Fig. I(u). All other peaks came from the dcrivatization process.
We gratefully acknowledge the support given by the 131-itish
Diabetic Association towards the research.
I . Brownlee, M., Cerami. A. di Vlassara. H. ( I U X X ) N. f:ti<q/../. Alccl.
318,1315-1321
2. Wadman. S. K.. I)e I3ree. P. K.. Van Sprang, F.J.. Knmerling. J . I?.
Haverkarnp. J. di Vligenthart. J. F. C. ( 1 0 7 5 ) C ~ l i t r .('lrivi. i1i.m
5 9 , 3 13-320
3. Ahmed, M. U., Thorpe, S. K. & Baynes. J . W. (19x6) J. Hiol.
C%ivn. 261,4X8+4X94
4. Hofmann, K., Stutz. E., Spuhler, (i.. Yaiima, H. Kr Schwartz. E. '1..
(1960)J. Am. ('hem. So(,. 82, 3727-3732
5. Chin, C. C. Q. & Wold, F. ( 1975) Ardz. Hioc,licw~.Riopliyx 167.
44x3-45 1
6. Climie, I. J. G. & Evans, I). A. ( 19x2) 7i.rrcrlrcdrorr 38.007-7 I I
7. Ahmed, M. U., Dunn, J. A,. Walla, M . I).. Thorpe, S. K. Kr
Haynes, J. W. ( 10x8) J. Hiol. C'licm. 263, XX 16-XX2 I
Received 14 June 10x9
Effect of electromagnetic induction on impulse conduction in the frog nerve-muscle preparation
F. A. WALI* and A. I. J. BRAIN?
* Respirutoty Luhorutoty, The tlospitulfor Sick Children,
Great Ormond Street, London WC'IN, utid t Depurttnetit oJ
Anuesthetics, St Atidrews llospitul, London E3, U.K .
Both electrical ( E S )and magnetic stimulation have been used
o n excitable tissues, as a diagnostic and therapeutic aid [ 1 I.
T h e purpose of the present investigation was to study the
Abbreviations used: ES. electrical stimulation; LMI. electromagnetic induction.
effect of electromagnetic induction ( E M l ) on nerve impulse
conduction and muscle twitch contraction in the frog isolated
sciatic nerve-gastrocnemius muscle preparation, t o see if
EM1 inhibited neuromuscular transmission in this preparation.
T h e preparation was dissected and set up in an organ bath
containing 100 ml o f Ringer solution, at room temperature
(22-25°C). T h e nerve trunk was passed through an induction
coil (copper coil. 20- 120 turn, 0.5 mm diameter and 0.6 Q ).
T h e induction coil was wound o n a PVC tube (internal
diameter 2 mm). T h e nerve trunk was moistened with Ringer
lY89
1089
63 1st MEETING, GUILDFORD
solution. The gastrocnemius muscle was laid in the organ
bath, with one end pinned, while the other end was attached,
via a thread, to a force transducer leading to a pen recorder.
The contractile responses produced by ES or by EM1 were
recorded isometrically. The sciatic nerve was stimulated,
electrically, at 0.5 Hz with 0.25-0.6 V (supramaximal)and 1
ms duration. The pattern of ES was interrupted by a period
of EM1 (induced current from a d.c. source of 1.5-4 V, at a
frequency of 100 min-I), operated via a make-and-break
switch. The duration of the EM1 was 2-4 min. The results
showed that EM1 reduced and then blocked impulse conduction and the isometric twitch contractions [control
3.5 k 0.4 g tension, mean f s.E.M., n = 8 experiments (frogs)]
within 2 min of application of the EM1 to the sciatic nerve.
Recovery of the blocked twitches and impulse conduction (to
about 5.7 mV, nerve compound action potential) was
obtained within 4-5 min after the cessation of the EM1 and
the washing out of the preparation in control Ringer solution.
Electron microscopic (scanning)studies of the cross-sections
of the sciatic nerve showed that EM1 may produce lesions in
the nerve membrane, especially in the myelinated nerve
membrane (including the Schwann cells), e.g. producing
vacuoles and degeneration of the fibres. The mechanism of
this kind of inhibition by EM1 can be explained in terms of
an interruption of the ionic fluxes across the nerve
membrane (2-5), and production of lesions in the nerve
membrane, causing degeneration of the myelin sheath in the
frog sciatic nerve, and subsequent block of impulse conduction and neuromuscular transmission.
1. Buchtal, F. & Rosenfalck, A. ( 1966) Brain Res. 3, I - 122
2. Kolin, A. ( 1 968) IJhysics Today 21,39-50
3. Kolin, A,, Brill, N. Q. & Proberg, P. J. (1959) /'roc. Soc. Exp.
Biol. Med. 102,251-253
4. Oberg, P. A. (1973)Med. Biol. Eng. 1 1 , 5 5 5 6 4
5. Polson, M. J. R., Barker, A. T. & Freeston, 1. L. ( 1982) Med. B i d .
Eng. Comput. 20,242-244
Received 5 April 1989
Control of acetoacetate production from exogenous palmitoyl-CoA in isolated rat liver
mitochondria
presence of 2,4-dinitrophenol+ malonate. These conditions
inhibit entry of acetyl-CoA into the Krebs cycle and Bhydroxybutyrate production from acetoacetate [2, 17, IS].
We divided the system into two parts connected by an intermediate X (the acetyl-CoA:CoA ratio) (see [13, 191). We
considered CPT I, the carnitine carrier, CPT 11, the
enzymes of P-oxidation and of the electron transport chain
as a single step (beta) which results in the production of
Introduction
acetyl-CoA. The second step (acac) was the group of
enzymes which synthesize acetoacetate from acetyl-CoA (the
The regulation of carnitine palmitoyltransferase (CPT ) 1 ( E C
HMG-CoA pathway).
2.3.1.2 1 ) activity, by malonyl-CoA, is important in control of
The approach. We measured the rate of acetyl-CoA
ketogenic flux in livers of normal fed adult rats [ I ] .However, formation from palmitoyl-CoA, Jhe,,, (by measuring the rate
there is indirect evidence that significant control over keto- of oxygen consumption, Jo), the rate of acetoacetate producgenesis may be invested at other (intramitochondrial) sites tion from acetyl-CoA, J,,,,, and the concentrations of acetyl12-81.
CoA and CoA at three different acetyl-CoA:CoA ratios.
We have presented strong evidence that in addition to The ratios were set by addition of pyruvate, which is
CPT I, mitochondria1 3-hydroxy-3-methylglutaryl-CoA oxidized to acetyl-CoA by pyruvate dehydrogenase. Using
(HMG-CoA) synthase ( E C 4.1.3.5), the second enzyme of these data and the Flux Control Summation and Conthe HMG-CoA pathway, may exert control over the keto- nectivity Theorems (see (13, 19]),we calculated the overall
genic flux [ 9- 101. Changes in ketone body production occur
elasticities to the acetyl-CoA :CoA ratio and the overall flux
in parallel with changes in mitochondria1 HMG-CoA control coefficients over ketogenesis of the two defined comsynthase activity in liver mitochondria from adult and young ponents of the system.
rats [9-12]. These result from changes in the succinylation
Calculations. In the absence of added pyruvate (condition
(and inactivation) of the synthase and in the absolute
l), measured oxygen consumption is due to oxidation of the
amounts of enzyme present.
reducing equivalents released during conversion of
In this paper we use the 'top-down' approach [ 131of Meta- palmitoyl-CoA to acetyl-CoA [ 181:
bolic Control Theory [ 14-16] to investigate whether the
thus
enzymes of the HMG-CoA pathway can contribute sigJhel,l(
= 4/7 JO(
= J~icac(
(1)
nificantly t o control of ketogenesis. We question the frequent
assumption that CPT I is necessarily the only major control (where JhCtd(
1)and J,,,,( 1) are in units of nmol of acetyl-CoA
site.
produced or consumed/min per mg).
However, with added pyruvate (condition 2), oxygen consumption results from oxidation of reducing equivalents
Methods
from both palmitoyl-CoA and pyruvate, and Jhc,,(2)can be
7'hc~system. Our system consisted of isolated liver mitocalculated as follows:
chondria oxidizing palmitoyl-CoA to acetoacetate in the
PATTI A. QUANT,*t DANIELLE ROBIN,*
PIERRE ROBIN,* JEAN GIRARD* and
MARTIN D. BRAND?
*Centre de Recherches sur la Nutrition du C.N.R.S., 9 rue
Jules Hetzel, 92190 Meudon-Bellevue, France and
Department of Biochemistry, University of Cambridge,
Tennis Court Road, Cambridge CB2 1Q W, U.K.
Abbreviations uscd: CPT 1. carnitine palmitoyltransferase I (EC
2.3. I .2 I ); HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA.
tTo whom correspondence should be addressed at Department of
Clinical Biochemistry, University o f Cambridge, Addenbrooke's
Hospital, Cambridge CB2 2QK.
Vol. 17
To derive the overall elasticities of the two subsystems
(beta and acac) to X (the acetyl-CoA:CoA ratio), we plotted
Jheta
and J,,,, against the acetyl-CoA :CoA ratio (graph not
shown) and multiplied each slope by [X]/ZJ(with no
pyruvate).